SUMMARY/ABSTRACT (PROJECT 4 ? SYMONDS, CALIMMUNE CONSORTIUM) The few gene-based therapies for HIV-1 disease that have been tested in the clinic have been focused on protecting differentiated T-cells, principally through ablation or reduction of CCR5 expression. While several studies have proposed to mimic the natural situation by genetic modification of autologous HSPC to achieve knockdown/knockout of CCR5, we are the only group to test this approach in the clinic. Transplantation of such a gene modified stem cell provides two therapeutic benefits; 1) a new immune system would be formed from the repopulating cells, unimpaired by the detrimental effects of long term chronic HIV-1 infection, and 2) the newly repopulating cells would be protected from subsequent HIV-1 infection. However, unlike the ?Berlin? patient, complete bone marrow replacement is not likely to occur because of the risk of myeloablative conditioning and the low efficiency of engraftment of gene-modified HSPC in the absence of such conditioning. Therefore, in order to replicate the cure in a broader HIV-1 patient population, we must understand the factors that improve engraftment in HIV-1 disease and develop more effective and safer bone marrow transplant procedures. As indicated, current methods result in a relatively low level of repopulation, generally less than 1%. As a consequence, the engineered immune cells that are protected from HIV-1 are only present at low frequency and the vast majority of cells can still serve as targets and reservoirs for HIV-1 replication. We propose to address this limitation by assessing a combination therapy consisting of both a means to protect progeny cells from subsequent HIV-1 infection and to enhance stem cell engraftment and repopulation. Since we anticipate that even with greatly enhanced engraftment, there will still be residual non-gene modified cells that can replicate HIV-1, we will also include a chimeric antigen receptor (CAR) delivered through a peripheral T cell transplant to target those remaining cells that are not protected and become infected by HIV-1. The hypothesis to be tested in this project is that transplantation of gene-modified HSPC and T-cells will give rise to self-perpetuating cell populations that may provide a continuous means of controlling HIV and its pathogenic sequlae in HIV-positive individuals. In conjunction with the other projects, we propose to; 1) conduct safety and feasibility assessments that will guide the selection of lead therapeutic candidate(s); 2) produce GLP and GMP grade therapeutic lentiviral vector(s); 3) assess safety (pharmacology and toxicology) in an appropriate animal model; 4) qualify the transduction of human CD34+ HPSC and T cells, expansion of T cells, and formulation and cryopreservation of both cell populations; 5) assess the best patient population, entry criteria, mobilization and conditioning regimens, and trial design, and to develop a protocol for a Phase I safety and tolerability trial that will give us an early indication of in vivo efficacy, and 6) be ready to submit an IND for this trial by the end of the funding period.